微小RNA-20a对宫颈癌细胞迁移、侵袭、增殖及ATG7表达的影响

Influence of microRNA-20a on the migration,invasion,proliferation and ATG7 expression of cervical cancer cells

目的探讨微小RNA-20a(miR-20a)对宫颈癌Si Ha细胞迁移、侵袭和增殖的影响及可能机制。方法采用Lipofectamine 2000脂质体法将miR-20a抑制剂和阴性对照载体分别转染Si Ha细胞株。采用实时荧光定量PCR(QPCR)检测两组细胞中miR-20a的表达以验证转染效果,细胞迁移实验、Transwell细胞侵袭实验及MTT法分别检测miR-20a的表达抑制对Si Ha细胞迁移、侵袭和增殖的影响;荧光素酶报告实验及Western blotting验证miR-20a对下游预测靶基因自噬相关蛋白7(ATG7)的调控作用。结果 QPCR检测结果显示,与阴性对照组比较,miR-20a抑制剂组Si Ha细胞中miR-20a的表达量显著降低(P<0.01),证明转染模型构建成功。细胞迁移实验显示,转染48 h后,阴性对照组细胞的划痕愈合率为(75.68±5.94)%,高于抑制剂组的(38.24±7.68)%(P<0.01)。Transwell细胞侵袭实验显示,转染48 h后,阴性对照组穿膜细胞数为(96.35±10.23)个,多于抑制剂组的(23.54±7.26)个(P<0.01)。MTT法检测显示,阴性对照组和抑制剂组细胞增殖能力的差异无统计学意义(P>0.05)。与阴性对照组相比,转染miR-20a模拟物+ATG7野生型3’UTR报告基因载体的细胞荧光素酶活性显著下降(P<0.01),而转染miR-20a模拟物+ATG7突变型3’UTR报告基因载体的细胞荧光素酶活性无显著变化(P>0.05);Western blotting检测显示,与阴性对照组比较,抑制剂组细胞ATG7蛋白表达量显著增加(P<0.01)。结论降低miR-20a的表达能显著抑制宫颈癌细胞的迁移和侵袭能力,但细胞增殖能力不受影响,这可能与miR-20a直接作用于其下游靶基因ATG7并调控其表达紧密相关。

Objective To investigate the influence of microRNA-20 a（ miR-20 a） on regulating the invasion,migration and proliferation of cervival cancer Si Ha cells and the possible mechanism. Methods miR-20 a inhibitors and negative control vectors were transfected into cervival cancer Si Ha cells by using Lipofectamine 2000 liposome method. QPCR was used to detect the transfection effect of miR-20 a in the two groups of cells. Cell migration assay,transwell cell invasion assay and MTT assay were used to detect the effect of miR-20 a on the migration,invasion and proliferation of Si Ha cells,respectively. The effect of miR-20 a on the downstream target gene autophagy related protein 7（ ATG7） was verified using luciferase reporter assay and Western blotting method. Results QPCR showed that compared with negative control group,the expression of miR-20 a in inhibitor group was significantly reduced,showing that the transfection model was successfully constructed. Cell migration assay showed that at 48 h after transfection,wound healing rate of negative control group was（ 75. 68±5. 94） %,higher than（ 38. 24±7. 68） % of inhibitor group（ P〈0. 01）. Transwell cell invasion assay showed that the number of transmembrane cells in negative control group was 96. 35± 10. 23,more than 23. 54 ± 7. 26 of inhibitor group（ P〈0. 01）. MTT assay indicated that the difference of cell proliferation between both groups was not significant（ P〉0. 05）.Compared with negative control group,the luciferase activity in the group transfected with miR-20 a mimic and ATG7 wild-type 3＇UTR reporter gene vector was significantly decreased（ P〈0. 01）,while transfected with miR-20 a mimic and ATG7 mutant-type 3＇UTR reporter gene vector did not significantly change the luciferase activity（ P〉0. 05）. The results of Western blotting showed that the expression of ATG7 protein was increased in inhibitor group compared with negative control group（ P〈0. 01）. Conclusion Decreasing the expression of miR-20 a can significantly inhibit the migration and invasion of cervical cancer cells,but the proliferative ability of cells is not affected,which may be closely related to miR-20 a acting directly on its downstream target gene ATG7 and regulating its expression.